Thesis (Ph. D.)-- University of Washington, 2005. / Vita. Includes bibliographical references (leaves 126-142).

Chronic myeloid leukemia (CML) is characterized by clonal expansion of hematopoietic progenitor cells, result from the translocation (9:22). The oncogene BCR-ABL, in the Ph chromosome, is transcribed and translated into a fusion protein BCR / ABL. The ABL tyrosine kinase (TK) in the fusion protein is constitutively activated and is needed for the initial leukemogenic event of CML and its activity induces production of reactive oxygen species (ROS). Of particular relevance to CML is the fact that an increase of ROS can have consequences, facilitating genomic instability may contribute to disease progression. The aim of this study was to determine the oxidative status in patients with CML, in attendance at a university hospital (HUWC). This is a cross-sectional study consisted of 30 adult patients of both sexes with clinical and laboratory diagnosis of CML on treatment with inhibitors (TK) 1st and 2nd generation. The concentrations of malondialdehyde (MDA) and nitrite (NO2-) were performed by spectrophotometric method. The activities of enzymes glutathione peroxidase (GSH-Px) and catalase (CAT) were determined in hemolysate by Glutathione Peroxidase Cellular Activity Kit  Assay (Sigma-Aldrich) and spectrophotometry, respectively. Total glutathione, reduced glutathione (reduced GSH), glutathione (GSSG) were determined by Total Glutathione Activity Kit  (Assay Designs, Inc) and calculated the ratio GSH / GSSG. For statistical analysis of nonparametric data was used  and ANOVA test for multiple comparisons Tukey. It was considered the minimum level of significance of 5%. The average concentrations of NO2- and MDA were increased in CML patients compared to control, regardless of disease activity. The antioxidant profile was characterized by decreased CAT and GSH-Px increased also independent of disease activity. The reduced GSH is presented decreased, the GSSH, increased and GSH / GSSG decreased. It was observed that patients using protease inhibitors of TK 2nd generation of oxidative stress parameters were significantly elevated compared to controls. In the analysis of patients on imatinib were not detected significant changes in oxidative status. We conclude that patients with CML are under oxidative stress and impaired antioxidant activity. / A Leucemia mielÃide crÃnica (LMC) à caracterizada pela expansÃo clonal de cÃlulas progenitoras hematopoÃticas, resultante da translocaÃÃo (9:22). O oncogene de fusÃo BCR-ABL, no cromossomo Ph, à transcrito e traduzido numa proteÃna de fusÃo BCR/ABL. A tirosina quinase (TK) ABL na proteÃna de fusÃo à constitutivamente ativada sendo necessÃria para os eventos leucemogÃnicos iniciais da LMC e sua atividade induz a produÃÃo de espÃcies reativas de oxigÃnio (EROs). De particular relevÃncia para LMC à o fato de que um aumento de EROs pode ter consequÃncias, facilitando a instabilidade genÃmica podendo contribuir para a progressÃo da doenÃa. O objetivo do estudo foi determinar o perfil oxidativo em pacientes com LMC, em acompanhamento ambulatorial no Hospital UniversitÃrio Walter CantÃdio (HUWC). Trata-se de um estudo transversal constituÃdo de 30 pacientes adultos, com diagnostico clÃnico e laboratorial de LMC, em tratamento com inibidores de (TK) de 1 e 2 geraÃÃo. As concentraÃÃes de malonaldeÃdo (MDA) e de nitrito (NO2-) foram realizadas por mÃtodo espectofotomÃtrico. As atividades das enzimas Glutationa peroxidase (GSH-Px) e catalase (CAT) foram determinadas no hemolisado, por kit Glutathione Peroxidase Cellular Activity Assay (Sigma-Aldrich) e por espectofotometria, respectivamente. Glutationa total, glutationa reduzida (GSH reduzida), glutationa oxidada (GSSG) foram determinadas por kit Total Glutathione Activity (Assay Designs, Inc) e calculada a relaÃÃo GSH/GSSG. Para a anÃlise estatÃstica de dados nÃo paramÃtricos foi utilizado o ANOVA e o teste de mÃltiplas comparaÃÃes de Tukey. Foi considerado o nÃvel mÃnimo de significÃncia de 5%. As concentraÃÃes mÃdia de MDA e de NO2- foram aumentadas nos pacientes com LMC em relaÃÃo ao controle, independente da atividade da doenÃa. O perfil antioxidante foi caracterizado pela diminuiÃÃo da CAT e aumento da GSH-Px tambÃm independente da atividade da doenÃa. A GSH reduzida se apresentou diminuÃda, a GSSH, aumentada e a relaÃÃo GSH/GSSG diminuÃda. Os pacientes em uso de inibidores de TK de 2 geraÃÃo apresentaram parÃmetros do estresse oxidativo significativamente elevados em relaÃÃo ao grupo controle. Conclui-se que os pacientes com LMC estÃo sob estresse oxidativo e com atividade antioxidante comprometida.

Le but des travaux est de caractériser l’influence de la signalisation VEGF, en particulier la signalisation autocrine VEGF, sur la biologie et la sensibilité/résistance aux médicaments anticancéreux de cellules de cancer colorectal. Nous avons souhaité caractériser l’impact de la signalisation autocrine VEGF dans des modèles de CRC avec une résistance naturelle au bevacizumab, un anticorps anti-VEGF. Bien que ce composé soit actif dans le CRC, une sous population de patients ne répondent pas au traitement. Nos résultats montrent une sur régulation de la voie autocrine HIF-VEGF-VEGFR en réponse à une exposition prolongée au bevacizumab dans les cellules bevacizumab résistantes. Si la résistance à cet anticorps est bien établie, d’autres inhibiteur de la voie VEGF restent actifs (comme la petite molécule ciblée nintedanib) et peuvent inhiber la voie mTOR. La signalisation VEGF autocrine joue un rôle dans la survie de cellules de CRC. Chez les sujets résistants au bevacizumab, il serait intéressant d’introduire le nintedanib seul ou en combinaison pour accentuer l’inhibition angiogénique. Une autre combinaison d’agents (anti VEGF(R) et anti EGFR) a montré une efficacité dans des modèles de CRC en préclinique. La combinaison du bevacizumab et d’une petite molécule ciblant EGFR (erlotinib) a montré une plus grande efficacité que le bevacizumab seul dans des modèles de CRC indépendamment du statut KRAS. Le bevacizumab induit une activation de la voie de survie EGFR dans les cellules tumorales et dans les cellules endothéliales associées à la tumeur. Cette activation se trouve diminuée avec l’introduction de l’erlotinib. Les résultats indiquent que la combinaison du bevacizumab et de l’erlotinib sont plus actif en thérapie de maintenance que le bevacizumab seul, même pour les patients mutés KRAS. Ces résultats ont mené à l’étude clinique positive GERCOR phase III DREAM dans le cancer du côlon métastatique. / The aim of the work is to characterize the influence of VEGF signaling , especially autocrine VEGF signaling , the biology and susceptibility / resistance to anticancer drugs of colorectal cancer cells. We wished to characterize the impact of the autocrine VEGF signaling in CRC models with natural resistance to bevacizumab , an anti -VEGF antibody. Although this compound is active in the CRC, a subpopulation of patients do not respond to treatment. Our results show an autocrine regulatory pathway HIF- VEGF- VEGFR in response to prolonged exposure to bevacizumab in bevacizumab resistant cells. If the resistance to the antibody is established, other inhibitos of VEGF pathway remain active (such as small molecule nintedanib ) and can inhibit the mTOR pathway. Autocrine VEGF signaling plays a role in CRC cell survival. In subjects resistant to bevacizumab, it would be interesting to introduce the nintedanib alone or in combination to enhance the angiogenic inhibition. Another combination of targeted agents ( anti-VEGF (R) and anti EGFR) has shown efficacy in preclinical models of CRC. The combination of bevacizumab and a small molecule targeting EGFR (erlotinib) showed greater efficacy than bevacizumab alone in CRC models regardless of KRAS status. Bevacizumab induces activation of the EGFR survival pathway in tumor cells and in endothelial cells associated with the tumor. This activation is decreased with the introduction of erlotinib. The results indicate that the combination of bevacizumab and erlotinib are more active in maintenance therapy than bevacizumab alone, even for patients mutated KRAS . These findings led to the positive Phase III clinical study GERCOR DREAM in metastatic colon cancer.

Les DYRKs (dual specificity, tyrosine phosphorylation regulated Kinases) et CLKs (cdc2-like kinases) sont deux familles de kinases du groupe CMGC. Elles sont impliquées dans le développement de la maladie d'Alzheimer et de la trisomie 21. Nous présentons ici une optimisation et une caractérisation biologique détaillée des Leucettines, une famille d’inhibiteurs pharmacologiques de DYRKs/CLKs dérivés de la Leucettamine B, un alcaloïde extrait d'une éponge marine. Nous avons étudié la relation structure/activité de cette classe d'inhibiteurs sur un ensemble de réponses biologiques. Afin d'étudier les cibles potentielles de ces inhibiteurs, nous avons mis en œuvre une méthode de chromatographie d’affinité. La sélectivité de la Leucettine L41, sélectionnée comme représentative des Leucettines, a été étudiée par des essais d'activité et d'interaction de kinases recombinantes in vitro, et des tests de chromatographie d'affinité (Leucettines immobilisées sur billes d'agarose, compétition sur billes d’inhibiteurs non sélectifs). Des approches transcriptomiques et protéomiques ont été utilisées afin de mieux comprendre le mécanisme d'action cellulaire de la Leucettine L41. Ces approches ont confirmé la sélectivité de la Leucettine L41 pour DYRKs et CLKs mais aussi révélé l’existence de cibles secondaires intéressantes. La Leucettine L41 module l'épissage alternatif de pré-ARNm. Elle présente des propriétés neuroprotectrices vis-à-vis de la mort cellulaire induite par le glutamate. Les Leucettines méritent un développement en tant qu'agents thérapeutiques potentiels pour le traitement de la maladie d'Alzheimer et de la trisomie 21. / DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) are two families of kinases belonging to the CMGC group. They are involved in the development of Alzheimer's disease and Down syndrome. We here present the optimization and a detailed biological characterization of Leucettines, a family of pharmacological inhibitors of DYRKs/CLKs derived from Leucettamine B, an alkaloid produced by a marine sponge. We studied the structure/activity relationship of this class of inhibitors on a set of biological responses. To investigate potential targets of these inhibitors, we implemented an affinity chromatography method. The selectivity of Leucettine L41, selected as a representative Leucettine, was studied by in vitro activity and interaction assays of recombinant kinases and affinity chromatography approaches (Leucettines immobilized on agarose beads, competition on non-selective inhibitors). Transcriptomics and proteomics approaches were used to better understand the cellular mechanism of action of Leucettine L41. These approaches confirmed the selectivity of Leucettine L41 for DYRKs and CLKs but also revealed the existence of interesting secondary targets. Leucettine L41 modulates alternative splicing of pre-mRNAs. It displays neuroprotective properties towards glutamate-induced cell death. Leucettines deserve further development as potential therapeutic agents for the treatment of Alzheimer's disease and Down syndrome.

Le pazopanib est un inhibiteur de tyrosine kinase (ITK) multicible indiqué dans le traitement de cancers du rein métastatiques et de sarcomes des tissus mous métastatiques. Comme les autres ITK, il présente une forte variabilité pharmacocinétique (PK) interindividuelle et ses relations pharmacocinétiques-pharmacodynamiques (PK/PD) en font un candidat de choix pour un suivi thérapeutique pharmacologique, permettant de proposer une dose optimale adaptée à chaque patient. La pratique clinique s'oriente vers une utilisation des ITK en association avec d'autres médicaments anti-cancéreux afin de potentialiser l'activité anti-tumorale. Ainsi, les études de phase I en association prévoient des explorations PK permettant d'évaluer l'existence d'éventuelles interactions PK entre les médicament(s) associé(s). Les données PK issues de deux études de phase I impliquant le pazopanib en association soit avec le bevacizumab, soit avec le cisplatine, ont été analysées. L'analyse PK par méthodologie de PK de population a confirmé la forte variabilité PK interindividuelle du pazopanib et a permis d'identifier les différentes sources de variabilités : inter-, intraindividuelle et variabilité due à une interaction médicamenteuse. Le pazopanib est très fortement lié aux protéines plasmatiques (>99%). L'étude de ses concentrations libres permet d'avoir accès aux concentrations pharmacologiquement actives. Ainsi, une méthode de dialyse à l'équilibre couplée à un dosage par UPLC/MS-MS a été mise au point afin de déterminer la fraction libre plasmatique du pazopanib in vitro et ex vivo. Les relations PK/PD entre concentrations libres et survenue de toxicités par rapport aux concentrations totales ont été étudiées / Pazopanib is a multitarget tyrosine kinase inhibitor (TKI) approved for the treatment of advanced renal cell carcinoma and metastatic soft tissue sarcoma. Like most TKI, pazopanib exhibits a large inter-individual pharmacokinetic (PK) variability, with a 1:8 plasmatic exposure variation factor between individuals. Pharmacokinetic/Pharmacodynamic (PK/PD) relationships suggest that therapeutic drug monitoring would be useful for individualized dosing of this drug. In current clinical practice, TKI are often combined with other anticancer drugs in order to optimise anti-tumour activity. However, therapeutic combinations can potentially lead to drug interactions. Therefore, PK investigations are needed in order to evaluate any PK interaction between each compound. PK data from two different phase I studies evaluating pazopanib in combination either with a cytotoxic, cisplatin, or with another targeted therapy, bevacizumab, were analysed. PK analysis of both studies using a population PK approach, confirmed the high inter-individual PK variability of pazopanib and allowed us to quantify different sources of variability: inter-, intra- patient variability and variability due to drug-drug interactions. Pazopanib is very highly bound to plasma proteins (>99%). Unbound drug concentrations in plasma are thought to be a more reliable indicator of the therapeutic target. Therefore equilibrium dialysis method coupled with UPLC/MS-MS assay has been optimized for the determination of plasma unbound fraction of pazopanib in vitro and ex vivo. PK/PD relationships between unbound plasma concentrations and toxicity in comparison with total plasma concentrations were studied for pazopanib.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Cancer is a global health catastrophe, with neuroblastoma, the most common solid childhood tumor, and glioblastoma, a deadly brain tumor, being aggressive and unresponsive to current treatment modalities. These tumors are known to utilize uncontrollable cell proliferative capabilities as a mechanism for tumor survival. Therefore, malignant cell growth can be mitigated by targeting the essential proteins that regulate cell growth, such as receptor tyrosine kinases (RTKs). Under normal physiological conditions, RTKs bind with varying affinity to mitogenic stimuli such as growth factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) which, in turn, leads to receptor phosphorylation and activation.

The cells and molecules that comprise the immune system are essential for mounting an effective response against microbes. A successful immune response limits pathology within the host while simultaneously eliminating the pathogen. The key to this delicate balance is the correct recognition of the pathogen and the appropriate response of immune cells. Cellular activation originates through receptors that relay information about the state of the microenvironment to different compartments within the cell. The rapid relay of information is called signal transduction and employs a network of signaling mediators such as kinases, phosphatases, adaptor molecules, and transcription factors. IL-2 inducible T cell kinase (Itk) is a non-receptor tyrosine kinase that is an integral component of signal transduction downstream of many immunoreceptors. This dissertation describes two distinct pathways that utilize Itk in both phases of the immune response. T cells use the TCR to sense a multitude of peptide-based ligands and to transmit signals inside the cell to activate cellular function. In this regard, the diversity of ligands the T cells encounter can be portrayed as analog inputs. Once a critical threshold is met, signaling events transpire in close proximity to the plasma membrane to activate major downstream pathways in the cell. The majority of these pathways are digital in nature resulting in the on or off activation of T cells. We find, however, that altering the TCR signal strength that a T cell receives can result in an analog-based response. Here, the graded expression of a transcription factor, IRF4, is modulated through the activity of Itk. We link this graded response to an NFAT-mediated pathway in which the digital vs. analog nature has been previously uncharacterized. Finally, we demonstrate that the repercussions of an analog signaling pathway is the altered expression of a second transcription factor, Eomes, which is important in the differentiation and function of T cells. These results suggest that Itk is crucial in the modulation of TCR signal strength. Mast cells primarily rely on the IgE-bound FcεR1 for pathogen recognition. Crosslinking this receptor activates mast cells and results in degranulation and cytokine production via an expansive signaling cascade. Upon stimulation, Itk is recruited to the plasma membrane and phosphorylated. Little else is known about how Itk operates inside of mast cells. We find that mast cells lacking Itk are hyperresponsive to FcεR1-mediated activation. This is most apparent in the amount of IL-4 and IL-13 produced in comparison to wild-type mast cells. Increased cytokine production was accompanied by elevated and sustained signaling downstream of the FcεR1. Finally, biochemical evidence demonstrates that Itk is part of an inhibitory complex containing the phosphatase SHIP-1. These results indicate a novel function for Itk as a negative regulator in FcεR1- mediated mast cell activation.

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